Composition forstimulating bone growth and differentiation and method for isolating same
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example 1
Cell Culture and Radiolabelling
[0177]Bone precursor MC3T3 cells were grown in 250 ml tissue culture flasks in 5% FCS / DMEM in a 10% CO2 / air-humidified incubator. When isolating logarithmic growth HS, radiolabel was added 24 h post-passaging and the cells allowed to grow unhindered for 3 days. To isolate HS from contact-inhibited cells, media on the cells was changed to 0.5% FCS / DMEM post-confluence and radiolabelled (20 μCi / ml) 24 h after the media was changed. Cells were maintained at confluence for 3 days and then the media collected and frozen at −20° C. until required. Cell membranes were prepared in lysis buffer (1% Triton X100, 150 mM NaCl, 10 mM Tris pH 7.4, 2 mM EDTA, 0.5% NP 40, 0.1% SDS containing the protease inhibitors 1 mM sodium orthovanadate, 10 μg / ml leupeptin, 1 μg / ml aprotinin and 1 mM PMSF). The cellular ECM was collected with lysis buffer plus 6 M Urea.
example 2
Determination of Metabolic Activity Using WST-1
[0178]Unless otherwise indicated, MC3T3-E1 cells were plated at 5000 cells / cm2 into wells of a 96 well plate in triplicate, allocating 3 wells to each time point, and grown in osteogenic media for 3-10 days. The Cell Proliferation Reagent WST-1 (Roche Diagnostics, Singapore) was added to triplicate wells at each time point, diluted 1:10 into the media. The reaction was catalysed by the conversion of WST-1, a tetrazolium salt, into formazon by mitochondrial dehydrogenase, which directly correlates to the number of metabolically-active cells in the culture. The reaction is incubated for 37° C. for 30 min, liberating a red colour, and read at 450 nm with a reference wavelength of 630 nm on a Victor3™ Multilevel Plate Reader (Perkin Elmer, Boston, Mass., USA). A blank well containing only media was used for background correction due to discolouration by the media.
[0179]As the assay can be performed and read under sterile conditions, cells f...
example 3
Determination of Cell Proliferation Using BrdU
[0180]Cell proliferation was analysed with a Cell Proliferation ELISA colorimetric kit (Roche, Switzerland). MC3T3-E1 cells were incubated with 10 μM BrdU for 2 h at 37° C., denatured, fixed and incubated with anti-BrdU-POD for 90 min at RTP according to the manufacturer's instructions. The reaction was catalysed by the addition of a tetramethylbenzidine substrate solution and terminated after 15 min with 1 M H2SO4. The absorbance was read at 450 nm (with a reference of 690 nm) using a Bio-Rad® Benchmark™ Microplate Reader (Bio-Rad, CA, USA) and corrected using blank and background controls.
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